Abstract Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a decimating infectious disease affecting one third of the human population and causing around two million deaths every year. Standard TB treatment uses a combination of different antibiotics that target a number of metabolic processes, RNA and cell wall synthesis, and energy metabolism in mycobacteria resulting in bactericidal action. The limited effectiveness of current treatments is largely due to their lengthy (>6 months) and complex nature, which leads to poor compliance from patients. The emergence of multi-drug resistant (MDR) TB and of the virtually untreatable extensively resistant (XDR) TB has heightened the need for new targets and innovative strategies to tackle TB infections. Mycobacterium protein tyrosine phosphatase B, mPTPB, is a bacterial virulence factor that is restricted to the species that causes TB in humans. In addition, deletion of mPTPB impairs the ability of M. tuberculosis to survive in macrophages and animals. Consequently, mPTPB represents an attractive target for novel therapeutics against MDR and XDR TB. Specific inhibition of mPTPB could provide a novel means of therapy with minimal side effects on the host, given the phylogenetic distance between M. tuberculosis and human phosphatases. Moreover, since mPTPB is secreted into the cytosol of host macrophages, drugs targeting mPTPB are not required to penetrate the waxy mycobacterial cell wall. The goal of this application is to target mPTPB for new and improved anti-TB agents. To achieve our stated goal, a collaborative and multidisciplinary research program is designed to identify and characterize potent and selective small molecule mPTPB inhibitors and to evaluate their mechanism of action and potential to be used as new therapeutics for drug resistant TB. Structure-guided combinatorial synthesis will be used to generate bicyclic salicylic acid compounds that are sufficiently polar to bind the mPTPB active site, yet remain capable of efficiently crossing cell membranes. High throughput screening enables rapid evaluation and characterization of the efficacy of the library compounds as mPTPB inhibitors. X-ray crystallographic structural studies of mPTPB in complex with selected lead compounds will reveal the molecular basis of mPTPB inhibition, and provide a structural framework for further mPTPB inhibitor optimization. Specific, high-affinity mPTPB inhibitors identified in this program will be evaluated for their activity against M. tuberculosis in both macrophages as well as in animal model of TB infection in order to determine the preclinical potential of mPTPB inhibitors. These mPTPB inhibitors will not only have promise as medicinal agents to combat MDR and XDR TB but also function as research tools for elucidating the roles of mPTPB in host signaling pathways involved in the pathogenesis of M. tuberculosis. Finally, substrate-trapping and proteomic approaches will be employed to identify mPTPB substrates in order to understand the mechanism by which mPTPB mediates mycobacterial survival in the host.